Inflammation, a basic host defensive response, is crucial for resistance to injury, infectious agents or other noxious stimuli. Nevertheless, excessive and persistent inflammation often leads to various chronic diseases such as cardiovascular, osteoporosis, diabetes, obesity and gastro-intestinal inflammatory diseases. According to WHO, chronic diseases are the leading cause of morbidity and mortality both in developed and developing countries, and represent 60% of all deaths in the world. This figure rises to 87% in Europe and it is expected that more people will be affected by chronic diseases over the next few decades. Increased treatment-related health care costs have made chronic diseases a real health problem to the societies and the concern to prevent or treat these illnesses through diet has increased.
Daily diet is comprised of variety of nutrients including proteins. Bioactive peptides derived from dietary proteins particularly milk may modulate different body functions both at intestinal and systemic levels and ultimately contribute in maintaining consumer health. It has been shown that casein hydrolysates or peptides present in them exhibit anti-inflammatory activity by inhibiting and/or reducing the expression of inflammatory markers and/or by modulating their activity. For example, besides the fragment 106-169 of the bovine κ-casein, anti-inflammatory activity was demonstrated for 84VPP86 and 74IPP76 peptides from the bovine β-casein which reduce in vivo the mRNA expression of inflammatory cytokines (IL-6 and IL-1β). Three ways for releasing peptides from native proteins can be: (i) in vitro enzymatic hydrolysis, (ii) during gastrointestinal digestion or (iii) during fermentation by lactic acid bacteria.
Streptococcus thermophilus, a lactic acid bacterium previously known for conferring organoleptic properties to dairy products, has also been shown to generate bioactive peptides from milk proteins through its cell envelope proteinase (PrtS), which is anchored to cell wall by the transpeptidase sortase SrtA. However, during fermentation the pH decreases due to production of lactic acid and consequently the activity of PrtS is reduced or inhibited (optimal activity at pH 7.5), resulting in lower peptide contents. Therefore, to overcome this problem, S. thermophilus LMD-9-delta-srtA mutant strain was constructed to release in growth medium PrtS, which after purification was used to hydrolyze a caseinate to produce a hydrolysate with higher peptide content, which was afterward fractionated.
The main goal of the study was to evaluate the immunomodulatory potential of the peptide fractions obtained after fractionation by ultrafiltration and diafiltration of a hydrolysate resulting from the hydrolysis of a caseinate by PrtS purified from the growth culture of S. thermophilus LMD-9-delta-srtA strain, using peripheral blood mononuclear cells (PBMC) from 4 human donors.
Materials and Methods:
S. thermophilus LMD-9-delta-srtA strain was cultured for 8 h in yeast-lactose (YL) medium to obtain PrtS-rich supernatant. Followed by batch chromatography using diethylaminoethyl (DEAE) cellulose DE23 (Pharmacia, Uppsala, Sweden) resin and discontinuous gradient of NaCl, PrtS-rich fraction was concentrated by ultrafiltration using Amicon® Ultrafiltration system (cutoff threshold 50 kDa, Milipore, Jaffery, USA). The concentrated PrtS-rich fraction was then used to hydrolyze an industrial caseinate comprising of 87% proteins (92% casein and 8% whey proteins). This initial hydrolyzate (F1) was further fractionated by ultrafiltration using a 3 kDa cut-off membrane to isolate non-hydrolysed proteins in the form of retentate (F2) and peptide fraction as permeate (F3). The F3 fraction was then concentrated by ultrafiltration and diafiltration using a 1 kDa cutoff membrane to obtain retentate (F4) and permeate (F5). The peptide fractions were co-incubated in vitro with PBMCs (Clinisciences, Nanterre, France) and secretion of the IL-10 anti-inflammatory cytokine and of the IL-12 pro-inflammatory cytokine was measured in the cellular medium. The IL-10/IL-12 ratio makes it possible to evaluate the immunomodulatory potential of the peptide fractions.
Using DEAE cellulose DE23 batch chromatography with NaCl gradient, the PrtS was predominantly recovered in the fraction eluted by 0.4 M NaCl since about 90% of the initial activity was found in this fraction. Hydrolysis of caseinate by PrtS was not total since size-exclusion chromatography showed that a significant proportion of proteins remained unhydrolyzed under the conditions used. The protein concentration of the hydrolyzate was about 5 g/L with about 1.6 g/L of peptides. F4 fraction containing peptides whose molecular mass was between 1000 and 3000 Da was free of any salts contrary to F3 and F5 fractions. Anti-inflammatory activity of the five peptide fractions was evaluated by quantifying IL-10 and IL-12 production in vitro after co-incubating individually with PBMCs obtained from four healthy donors who did not use anti-inflammatory drugs for a significant period of time. All the fractions tested at concentrations of 0.2 and 1.0 mg of protein matter / mL led to a secretion of IL-10 by PBMCs except F3 and F5 where the secretion of IL-10 was observed only for concentration of 0.2 mg/mL. The absence of secretion with these fractions tested at 1.0 mg/mL was probably due to an excess of salt content. However, F4 fraction induced higher level of IL-10 production (between 70 and 400 pg/mL according to the PBMC donor) followed by F2 fraction regardless of concentration used. No IL-10 was detected when PBS alone was applied. Similarly, none of the peptide fraction favored the release of the pro-inflammatory cytokine IL-12. Results obtained with the cells of all the donors gave the same conclusions.
PBMC treated with F4 fraction, among all peptide fractions, secreted higher levels of IL-10 in vitro and, therefore, this fraction displayed potential anti-inflammatory activity, as it did not trigger any secretion of IL-12. This peptide fraction is rich in peptides with molecular mass ranging between 1 and 3 kDa. Hence, milk proteins represent a promising source of peptides with potential anti-inflammatory activity and these peptides could be released directly by S. thermophilus or by its protease used as a biotechnological tool. It could be interesting to identify the peptide sequences of the F4 fraction in order to characterize it with the aim of using it in the development of functional fermented milk products.